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An avian influenza virus, A/turkey/England/50-92/91 (H5N1), showed extremely high virulence in chickens, although its hemagglutinin (HA) cleavage site sequence (R-K-R-K-T-R), having a nonbasic (Thr) residue at the second position (P-2) from the carboxyl terminus of HA1, does not conform to the previously established consensus sequence motif, X-X-R/K-X-R/K-R (X = nonbasic residue), for highly virulent phenotype of the H5 virus. When we evaluated the HA cleavability of this strain in chicken embryo fibroblast culture, we observed that, unlike other HAs with a Thr residue at P-2, this HA was efficiently cleaved. These findings suggest that a nonbasic residue at the P-2 does not affect its recognition and catalyzation by cleavage enzymes that are otherwise influenced by steric structure around the cleavage site.

Degenerate oligonucleotide primers were synthesized to amplify nucleotide sequences from portions of the fusion protein and matrix protein genes of Newcastle disease virus (NDV) genomic RNA that could be used diagnostically. These primers were used in a single-tube reverse transcription PCR of NDV genomic RNA coupled to direct nucleotide sequencing of the amplified product to characterize more than 30 NDV isolates. In agreement with previous reports, differences in the fusion protein cleavage sequence that correlated genotypically with virulence among various NDV pathotypes were detected. By using sequences generated from the matrix protein gene coding for the nuclear localization signal, lentogenic viruses were again grouped phylogenetically separate from other pathotypes. These techniques were applied to compare neurotropic velogenic viruses isolated from an outbreak of Newcastle disease in cormorants and turkeys. Cormorant NDV isolates and an NDV isolate from an infected turkey flock in North Dakota had the fusion protein cleavage sequence 109SRGRRQKRFVG119. The R-for-G substitution at position 110 may be unique for the cormorant-type isolates. Although the amino acid sequences from the fusion protein cleavage site were identical, nucleotide sequence data correlate the outbreak in turkeys to a cormorant virus isolate from Minnesota and not to a cormorant virus isolate from Michigan. On the basis of sequence information, the cormorant isolates are virulent viruses related to isolates of psittacine origin, possibly genotypically distinct from other velogenic NDV isolates. These techniques can be used reliably for Newcastle disease epidemiology and for prediction of pathotypes of NDV isolates without traditional live-bird inoculations.

Partial-length gene 4 cDNA encompassing the VP8* portion of VP4 from chicken rotavirus (RV) Ch-1, turkey RV Ty-1 and Ty-3, and pigeon RV PO-13 was sequenced and compared to RV 993/83 isolated from a calf with diarrhea. Ninety-six percent amino acid sequence identity was seen between VP8* from calf RV 993/83 and pigeon RV PO-13, while only 77 to 83% identity was seen in comparison with turkey and chicken RV VP8* sequences. Phylogenetic tree analysis places all avian RV (including calf isolate 993/83) on a branch at the bottom of the VP8* tree. When tested with three neutralizing monoclonal antibodies raised against pigeon RV PO-13, RV 993/83 and Ty-1 share three, Ch-1 shares two, and Ty-3 shares one neutralizing epitope(s) on VP4 with pigeon RV PO-13.

Two enzyme-linked immunosorbent assays (ELISA) developed in the authors' laboratory for turkey rhinotracheitis serological testing, a commercial ELISA kit, and two virus-neutralization (VN) assays were compared with respect to the efficiency of these assays for serological monitoring in specific-pathogen-free (SPF) turkeys inoculated with four pathogenic isolates of turkey rhinotracheitis virus, with or without previous live vaccination. Both the live vaccine and the different isolates of virus were shown to induce antibody rises, the detectability of which varied depending on the ELISA or VN assay used for serological testing. The results show that 3 weeks after vaccination with an attenuated strain, the choice of an inadequate antigen for serological testing may be the cause of an apparent lack of immunogenicity of the vaccine, and that 2 weeks after challenge, such a choice in ELISA can also hinder the early diagnosis of a TRT virus infection in both vaccinated and unvaccinated turkeys.

We analyzed the long terminal repeat (LTR) of the lymphoproliferative disease virus of turkeys for sequences that influence its promoter activity by using the chloramphenicol acetyltransferase assay. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 regulatory sequences were used for these studies. Through transfection experiments, we identified a negative regulatory element residing at the 5' end of the U3. The two imperfect direct repeats (DRs) located at nt - 170 to - 125 upstream of the RNA transcription site were identified as enhancer elements which could stimulate transcription of a heterologous promoter in an orientation independent manner. Specific interaction of nuclear factors with the DRs element was identified. The two DRs contain cArg motifs which are suggested to play a role in tissue specific expression of several cellular genes.

Two experiments were conducted to determine intestinal disaccharidase activity in 1-day-old commercial turkey poults inoculated with astrovirus. Small intestinal samples were collected on days 0.5, 1, 3, and 7 postinoculation (PI) in Expt. 1 and on days 7, 10, and 14 PI in Expt. 2 and evaluated for specific maltase activity (SMA). Astrovirus infection was verified on day 7 PI by immune electron microscopy of intestinal contents. Inoculated poults developed diarrhea and a transient, significant decrease in intestinal SMA. SMA was significantly (P < 0.05) lower in astrovirus-inoculated poults than in control poults throughout the entire small intestine from day 3 through day 7 PI. However, SMA had returned to normal in inoculated poults by day 10 PI and was significantly higher than control values (P < 0.05) in all sections of the small intestine, except in the proximal jejunum, by day 14 PI. Decreased SMA caused by astrovirus infection resulted in disaccharide maldigestion, malabsorption, and subsequent osmotic diarrhea. As astrovirus was cleared from the intestinal tract, SMA was restored and diarrhea was resolved.

In three separate experiments, 2- or 5-day-old commercial turkey poults were inoculated orally with astrovirus and examined for clinical signs and for gross and microscopic lesions over a period of 14 days. By day 2 postinoculation (PI), inoculated poults had developed diarrhea, generalized loss of intestinal tone, and dilated ceca that contained light-yellow fluid feces and gas; these changes persisted through day 10 PI. Mild crypt hyperplasia was the only change discernible by light microscopy, and it was first noted in the proximal jejunum on day 1 PI, in the distal jejunum and ileum on day 3 PI, and in the duodenum on day 5 PI. A significant (P < 0.05) increase in crypt depth and area was documented by image analysis on day 3 PI. Ultrastructural evaluation revealed intracytoplasmic aggregates of astrovirus in enterocytes on the sides and base of villi in the ileum and distal jejunum on day 3 PI. Based on the findings, it was concluded that astrovirus caused lesions and replicated in both upper and lower segments of the small intestine in turkey poults.

Tom turkeys were experimentally inoculated with eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus; semen was examined for presence of virus and ability to transmit infection by artificial insemination. Mild depression and inappetence were observed in tom turkeys inoculated with either EEE virus or HJ virus. Toms were viremic on days 1-2 postinoculation (PI), and virus was shed in semen on days 1-5 PI. Semen collected from EEE-virus-inoculated or HJ-virus-inoculated toms on days 1-2 PI and inseminated into turkey breeder hens transmitted the infection. EEE virus was detected in one of 10 hens after insemination with semen from EEE-virus-inoculated toms, and HJ virus was detected in three of 10 hens after insemination with semen from HJ-virus-inoculated toms. These results indicate that semen is a potential vehicle for transmission of EEE virus and HJ virus.

The DNA sequence of a 4.792-kb fragment comprising 3.176 kb of the long unique region (UL) and 1.605 kb of the internal repeat (IRL) flanking UL of herpesvirus of turkeys (HVT) was determined. Three potential open reading frames (ORFs) and an origin of replication have been identified. ORF 1, which maps entirely within UL, has the capacity to code for an 82K protein, 731 amino acids long, which has a counterpart in Marek's disease virus (MDV) but not in other herpesviruses. ORF 2 has the potential to encode a protein consisting of 129 amino acids with a predicted M(r) of 13.5K which appears to be unique to HVT. ORF 3 is encoded entirely within IRL and codes for an 84 amino acids long protein with a predicted M(r) of 8.5K. ORF 3 shows significant homology with the C-terminal region of the MDV-1-specific phosphoprotein pp38 and its recently identified homologue in MDV-2. Northern blot analysis of RNA extracted from HVT-infected chick embryo fibroblasts identified a 5.6-kb RNA transcribed in a leftward direction toward UL scanning ORF 1, ORF 2, and ORF 3 and a 2.8-kb also transcribed leftward toward UL which spanned only ORFs 2 and 3. In addition, a 2.3- to 2.8-kb RNA family was transcribed rightwards through the origin of replication. In vitro transcription and translation of ORF 1 and ORF 3 resulted in the synthesis of polypeptides consistent with their expected M(r), but ORF 2 failed to produce any translation product.

Although retroviral integration requires specific viral DNA sequences, factors which govern the choice of a chromosomal target site within an infected cell are less clear. For example, certain chromosomal regions may be inaccessible to the viral integration machinery, while others may favor integration. A recent paper by Withers-Ward et al.(1) addresses this issue using a polymerase chain reaction-based assay capable of identifying single integration events within a large population of infected cells. Their results show that integration can occur into many different chromosomal regions, and that local DNA structure can influence the site of integration within a given region.

The lymphoproliferative disease retrovirus (LPDV) induces an acute, horizontally transmitted disease of turkeys that is often fatal. Although LPDV cannot be grown in cultured cells, it was possible to isolate molecular clones of biologically active integrated proviral genomes from spleens of infected turkeys. Based upon molecular hybridization and nucleotide sequence comparisons of its pol gene, LPDV was shown to represent a distinct group of avian retroviruses most closely related to avian sarcoma-leukemia viruses. Here we report the complete nucleotide sequence of the LPDV genome as well as amino acid sequence analysis of its gag gene products. The genetic organization of LPDV is characteristic of members of the oncovirus subfamily. Further sequence comparisons of the gag gene confirmed that LPDV is most closely related to Rous sarcoma virus (RSV). However, the gag, pro, and pol open reading frames (ORFs) were in different translational phases so that the expression of their mature gene products would require the double frame-shifting mechanism utilized by simian retroviruses, mouse mammary tumor virus, and human T-cell leukemia virus. In contrast, the RSV proteinase is synthesized as part of the gag precursor. The LPDV gag gene differs from that of RSV as well as from all other retroviruses in that it encodes a unique 31,000-Da (p31) protein, located between the MA and the CA coding sequences. FOur short ORFs of unknown function were present, Whether the putative products of these ORFs account for the acute nature of LPDV-induced disease remains to be determined.

Nine open reading frames mapping in the short unique (US) region of the genome of herpesvirus of turkeys (HVT) were expressed by in vitro transcription and translation. The observed M(r)s of US10, SORF3 and US2 were as predicted from the sequence but there were discrepancies between the observed and predicted M(r)s of US1, protein kinase, gI, gD and gE. These could be accounted for in most cases by post-translational and co-translational processing. Analysis of the synthesized products at different time points provided evidence for post-translational modification of HVT protein kinase. Translation in the presence of microsomal membranes resulted in co-translational processing of HVT gD, gI and gE by glycosylation and signal peptide cleavage.

The increased use of serotype 2 Marek's disease virus (MDV) and serotype 3 turkey herpesvirus (HVT) as components of effective bivalent vaccines against Marek's disease (MD) prompted studies on the possible interactions of these two viruses in vitro and in vivo. The replication of the SB-1 strain of MDV was compared with replication of the FC126/2 strain of HVT in chickens and cell cultures infected with one or both viruses. Replication of MDV was reduced in the presence of HVT in both in vitro and in vivo systems. MDV plaque counts in dually infected chicken embryo fibroblast cultures inoculated with tissue-culture-propagated viruses were reduced by up to 91%; however, no inhibition was noted when inocula consisted of virus-infected buffy-coat cells. Plaque formation by MDV in chicken embryo fibroblast cultures was inhibited by virus-free conditioned medium from HVT-infected cultures. This conditioned medium also inhibited growth of vesicular stomatitis virus in a standard interferon assay. In chickens inoculated with both MDV and HVT, MDV viremia titers were lower and the dose required to infect 50% of susceptible chickens was increased 13-fold compared with chickens inoculated with MDV alone. In spite of these findings, there was no evidence that high concentrations of HVT interfered with either the ability of MDV to induce protective synergism in vivo or the protective efficacy of bivalent vaccines. No reciprocal inhibitory effects of MDV on the replication of HVT in vivo or in vitro were noted.

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to avian pneumovirus (APV) in chicken or turkey sera is described. The assay was capable of detecting serological responses as early as 11 days after chickens had been experimentally exposed to APV. The assay was evaluated by testing 4989 chicken or turkey sera from Canada (a known APV-negative country) and by testing 1190 chicken or turkey sera assumed positive from evidence of other laboratory results or clinical signs. This evaluation indicated that the ELISA was 98.7% sensitive and 99.5% specific. Evaluation of the agreement between the results of this ELISA and that of another laboratory was done by testing a panel of 218 chicken or turkey sera. The Kappa statistic for agreement was 0.92, indicating an excellent level of agreement between the two laboratories.

Turkey breeder hens were experimentally infected with strains of eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus previously isolated from turkey hens experiencing decreased egg production. Depression and inappetance were observed on day 1 postexposure (PE) in hens inoculated with either EEE virus or HJ virus, and egg production fell in each virus-inoculated group from approximately 75% to less than 20% within 2-3 days PE. Egg production remained depressed (less than 20%) for 15 days in EEE-virus-inoculated hens and for 7 days in HJ-virus-inoculated hens. EEE virus and HJ virus were recovered from various tissues on days 1-5 PE, and virus was detected in eggs laid on days 2-5 PE. The findings of this study confirm that EEE virus and HJ virus are potential causes of decreased egg production in turkey breeder hens.

Turkey poults from hens vaccinated against avian group A rotavirus were examined to study the transfer of maternally derived anti-rotavirus IgG (rIgG) to the mucosal surfaces (intestinal and tracheal), serum, yolk, and bile. During the first week of life, maternal rIgG titers in intestinal mucosal washings were 200-to-500-fold less than rIgG titers in the circulation, as determined by enzyme-linked immunosorbent assay (ELISA). The intestinal titers in 10- and 13-day-old poults were negligible. A moderate linear correlation (r = 0.6) was present between rIgG titer in the blood circulation and the intestines, with a serum cutoff level of 10,000 ELISA units. Maternal rIgG was detected in tracheal washings only during the first 3 days of life. Biliary rIgG titers were fourfold higher than intestinal titers at day of hatch but had declined considerably in 1-day-old poults. Yolk had relatively high rIgG titers at hatching. Maternal rIgG titer in the small intestine was determined after in situ ligation of the individual segments; it was highest in the duodenum, followed by the ileum and jejunum. There was evidence that rIgG in the intestine was transferred from the blood and not directly from the yolk sac. Bidirectional movement of rIgG between circulation and intestine was also detected. Maternal rIgG was not detected in the intestinal washings of progeny from hens naturally infected with rotavirus.

Tissue-culture-propagated marble spleen disease virus (MSDV-TC) and two preparations of spleen homogenate (MSDV-SH and MSDV-SH-TC) were compared as anti-hemorrhagic enteritis virus (HEV) vaccines in specific-pathogen-free turkeys. Both types of vaccines spread horizontally among turkeys, induced anti-HEV antibodies, and protected turkeys against challenge with virulent HEV. Antibody development and horizontal spread of virus occurred earlier in turkeys given MSDV-SH or MSDV-SH-TC than in those given MSDV-TC. Virulent HEV was serially passed in MDTC-RP19 cells. The 30th passage virus (HEV-P30) was nonpathogenic for turkeys but was immunogenic. Turkeys exposed to HEV-P30 had viral antigen in the spleen, developed neutralizing antibodies, and resisted virulent HEV. The principal difference between MSDV-TC and HEV-P30 vaccines was that MSDV-TC caused well-defined splenomegaly in turkeys, whereas HEV-P30 protected turkeys without causing spleen enlargement.

Wildlife are usually considered vectors, reservoirs or primary targets of infectious disease. A seldom considered epidemiological role which they can play involves their use as disease sentinels for the detection and monitoring of zoonoses. Their potential for such utilization has been demonstrated with the wild turkey (Meleagris gallopava intermedia) and St. Louis encephalitis in Texas and the white-tailed deer (Odocoileus virginianus) and California encephalitis in North America. The limitations and criteria which are important in the use of wild populations for "sentinel" duty are discussed.